Smad2/3/4 complex could undergo liquid liquid phase separation and induce apoptosis through TAT in hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-β signaling pathway may be associated with tumor progression. However, the relationship between TGF-β signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-β signaling pathway on HCC progression as well as the potential regulatory mechanism involved. Methods We conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni–NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo. Results We found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is −1032 to −1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis. Conclusion This study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-024-03353-x.

the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development.

Background
Hepatocellular carcinoma (HCC) is the most prevalent liver malignancy and is among the top leading causes of cancer-related mortality [1][2][3].It is reported that genetic mutations, chromosomal aberrations, molecular signaling pathways, and epigenetic deregulation have been implicated in HCC [4], but the mechanism of HCC development still remains ambiguity.Therefore, to reveal the mechanism of HCC development in a new sight is critical to solve this problem.
Smad2/3 and Smad4 assemble Smad2/3/4 complex then translocate to the nucleus and regulate target gene expression when TGF-β signaling is activated [10].Studies show that transcription factors can undergo LLPS with mediator to activate gene expression [15].Transcription coactivators, such as BRD4 and MED1, can also form phase-separated droplets to control gene expression [16].Smad2/3/4 complex is a well known transcription factor.In this study, we proved that Smad2/3/4 complex could undergo LLPS to active gene expression.
Phase separation takes part in some physiological activities [17][18][19].Such as cytoskeleton formation [20], RNA metabolism [21], nucleolar formation [22] and transcription [15,16].LLPS can reflect the highly dynamic changes of macromolecular substances in solution.The phase separation of protein and protein (or protein and nucleic acid) can form liquid droplets to activate or inhibit biochemical reactions.Recently, LLPS is reported to have relationship with pathologic process, for instance, rare genetic disease [23], neurodegenerative disease [17] and cancer [24].It is reported that LLPS can affect the progress of HCC.For example, LLPS of glycogen encapsulates YAP into glycogen droplets, inhibits the activation of Hippo pathway and drives the occurrence of HCC [24].The Twist1-YY1-p300 complex promotes miR-9 expression through LLPS, stimulates hepatoma cell invasion and metastasis [25].All these researches suggest that LLPS is related to HCC progress.In our research, we found that the LLPS of Smad2/3/4 complex could inhibit HCC progress.
Previous studies show that the disturbance of tyrosine metabolism is related to cancer progress [26].In addition, clinical patients with hereditary tyrosinemia are more likely to develop HCC [27,28].In HCC patients, serum tyrosine is often upregulated [29,30].These suggest that tyrosine metabolism imbalance is related to HCC process.The TAT gene encodes a mitochondrial protein tyrosine aminotransferase which is present in liver.TAT involves in tyrosine breakdown and converts tyrosine to p-hydroxyphenylpyruvate.In previous research, TAT acts as a tumor suppressive player in HCC [31].The regulatory mechanism of TAT expression still remains unknown.In our research, we found that Smad2/3/4 complex would undergo LLPS to regulate TAT expression.
In this study, we found that Smad2/3/4 may activate TAT expression through liquid liquid phase separation.The CCK-8 and colony formation assays results showed that Smad2/3/4 complex inhibited HCC progress was related to TAT.Western-Blot and Flow cytometry assays results suggested that Smad2/3/4 complex could active caspase-9 to improve hepatoma cell apoptosis through TAT.Our research further correlated TGF-β signaling pathway with apoptosis through

CCK-8 analysis
Transfect HUH7 and HepG2 cells with target vector via lip3000 (Followed the Reagent instructions).Inoculated cell suspension in 96-well plate (1000-5000 cells/well), Pre-incubate the plate in cell incubator at 37 ℃ according to experimental need.Add equal to 1/10 of the media volume CCK-8 (FC101-01, TransGen Biotech, Beijing, China) solution to each well and then incubate the plate in 37 ℃ for 1-3 h.Used a microplate reader to measure the absorbance at 450 nm.

ELISA analysis
Treated cells were lysed using RIPA buffer, tumors were frozen with liquid nitrogen and grind, then also lysed using RIPA buffer.Lysed cell and tumor was centrifuged at high speed and collected supernatant.The supernatant was used Human TyR ELISA Kit (CB15676-Hu, Shanghai Coibo Bio Technology, Shanghai, China) to test the tyrosine content according to the manufacturer's protocol.Using a microplate reader to measure the absorbance at 450 nm.

Recombinant protein expression and purify
We used E. coli to express recombinant protein (Tagged with EGFP/mCherry/EBFP), and protein (His tagged) was purified with Ni-NTA column.

Phase separation in vitro
Purified proteins were diluted in PBS, added PEG8000 at a final concentration 10% (M/V).The solution is expected to turn cloudy if the peptide is prone to liquid liquid phase separation.Use different NaCl concentration and temperature to test the environmental effect of liquid droplets formation.The liquid droplets were used detected by fluorescence microscope or laser confocal microscope.

Flow cytometry
Transfect HUH7 and HepG2 cells with vectors through lip3000, and knock down target genes by lentivirus (shRNA).To test hepatoma cell apoptosis, a total of 4 × 10 5 cells per sample were prepared as signal-cell suspensions of the treated cells, and stained using the Annexin V-FITC Apoptosis Detection Kit(Cat: 556547, BD Bioscience, USA).The cells were analyzed by flow cytometry.Raw data were analyzed through the FlowJo_ V10.8.1 software.

Tumor formation in nude mice
HUH7 cells well cultured in DMEM medium with 10% FBS and 1% penicillin streptomycin.Knock down of target genes by shRNA and screened at least 7d with puromycin.Pancreatic enzyme digestion the cells and count the numbers.Resuspend cells with the sodium chloride (0.9%).1.5-2 × 106 tredted cells were injected in one 6-8w nude mice.Start record the tumor growth when the tumor length grown to 3-5 mm.When tumor volume growth to 1000-1500 mm 3 , all mice were sacrificed and the tumor tissues were used for further study.

Cell culture
HUH7 and HepG2 cell lines were cultured in DMEM with 10% FBS and 1% Penicillin streptomycin.Environment with 5% CO 2 and hold on 37 ℃.Unless special note, most experiments of Smad2/3/4 complex activation were needed to add TGF-β1, the TGF-β1 concentration was 100-200PM.

TAT expression is related with Smad2 and Smad3 in HCC
In our research, we used the GEPIA database to analyze the expression levels of TAT in HCC patients.The results showed that TAT was differentially expressed in these two groups (Fig. 1A).Subsequently, a univariate COX prognostic analysis was performed, and TAT gene was identified as having a significant impact on survival prognosis in HCC, indicating that high expression of the TAT gene is associated with better survival prognosis (Fig. 1B).In addition, ROC curves were generated for 1, 3, and 5 years (Fig. 1C).Our data also showed that low mRNA expression of TAT in HCC samples was significantly associated with mild clinical stage and pathological grade (Fig. 1D, E).Furthermore, the mRNA levels of TAT were down-regulated in liver cancer tissues compared with paracancerous (Fig. 1F).These results suggest that TAT may play an inhibitory role in HCC progression.Previous research also has reported that mutation of TAT can advance HCC progression [31].However, the regulatory mechanism of TAT in HCC is unknown.
In our research, we observed a positive correlation between TAT mRNA expression and Smad2 and Smad3 in clinical HCC tissues (Fig. 1G, H).Taken together, our results indicate a tumor suppressive role of TAT in HCC progression, and a regulatory role of the TGF-β pathway in TAT expression.

TAT expression is regulated by Smad2/3/4 complex in HCC
When TGF-β signaling is activated, Smad2/3 and Smad4 assemble into the Smad2/3/4 complex, which then translocates to the nucleus to regulate target gene expression [10].In the aforementioned study, we observed a positive correlation between TAT expression and Smad2 and Smad3.Based on these findings, we postulated that the Smad2/3/4 complex might play a regulatory role in TAT expression in HCC cell lines.To further investigate this relationship, we successfully downregulated Smad2 using shRNA in HUH7 and HepG2 cell lines.Additionally, we overexpressed Smad2 in these cell lines (Supplementary Fig. S1A-D).QPCR and Western Blot analysis showed that knockdown of Smad2 downregulated the mRNA and protein expression of TAT in HUH7 and HepG2 cell lines (Fig. 2A-D).Further, we depressed and overexpressed Smad3 and Smad4, respectively (Supplementary Fig. S1E-L).The results showed that, the same as Smad2, Smad3 and Smad4 could also regulate TAT expression at both mRNA and protein level in HUH7 and HepG2 cell lines (Fig. 2E-L).These results suggest that TAT expression is regulated by Smad2/3/4 complex in hepatoma cells.

Smad2/3/4 complex undergoes liquid liquid phase separation
Smad2/3 and Smad4 assemble into a complex and translocate to the nucleus to regulate target gene expression when TGF-β signaling is activated [10].Previous studies have shown that transcription factors undergo phase separation to regulate gene expression, and proteins that are prone to this process contain specific domains [15].In particular, disorder sequences and PrLD domains are important for phase separation [17].We analyzed the sequence characteristics of Smad2, Smad3, and Smad4, and found that Smad2 and Smad3 had little disorder sequence and no PrLD domain, while Smad4 had a large amount of disorder sequence and a PrLD domain (Fig. 3A).These findings suggest that the Smad2/3/4 complex may undergo LLPS during gene regulation, and that Smad4 may drive this process.Further, we purified Smad4 (Tagged with EGFP) protein using E. coli.We observed that Smad4 formed Fig. 6 Knocking down Smad2, Smad3 and Smad4 could promote HCC progression.A Tumors from the nude mice to show the Smad2 effect on tumor progression.B Tumor growth curves of (A).C Tumors from the nude mice to show the Smad3 effect on tumor progression.D Tumor growth curves of (C).E Tumors from the nude mice to show the Smad4 effect on tumor progression.F Tumor growth curves of (D).G-I Tumor weight of each group, shSmad2 (G), shSmad3 (H), shSmad4 (I).J-L Tested TAT mRNA level of tumors from nude mice, shSmad2 (J), shSmad3 (K), shSmad4 (L).P < 0.05, *; P < 0.01,**; P < 0.001,***; P < 0.0001, **** (See figure on next page.)droplets in vitro (Fig. 3B).The formation of these droplets was affected by temperature, indicating that this was an example of a lower critical solution temperature phase diagram (Fig. 3C).NaCl concentration also influenced the formation of the droplets, suggesting that LLPS was driven by electrostatic interactions between the PrLD and disorder sequence (Fig. 3D).In vivo, we detected the formation of Smad4 liquid droplets form in HUH7 cells (Fig. 3E).To verify the liquid nature of these droplets, we conducted an experiment using fluorescent recovery after photobleaching (FRAP).The FRAP results revealed that the droplets exhibited rapid fluorescence recovery following bleaching (Fig. 3F), indicating that these droplets possessed liquid characteristics and indicating that Smad4 underwent liquid-liquid phase separation (LLPS).Additionally, we purified Smad2 and Smad3 using E. coli and found that the mixed Smad2/3/4 complex underwent phase separation to form liquid droplets in vitro (Fig. 3G).Collectively, these findings indicate that the Smad2/3/4 complex undergoes liquidliquid phase separation in Hepatoma cells.Previous studies have established that the Smad2/3/4 complex regulates target gene expression [10], and that LLPS of transcription factors is necessary for gene expression [15].Our research has found that the Smad2/3/4 complex undergoes phase separation, we believe that it also undergoes phase separation during transcription activity.

Smad2/3/4 complex binds to the TAT gene promotor site
Previous studies showed that Smad2/3/4 complex could bind to target gene promoter and increase promoter activity [32].In our research, we used bioinformatics analysis found that Smad2/3/4 complex might bind to the transcriptional sites of TAT (Fig. 4A), suggesting that Smad2/3/4 complex can regulate the expression of TAT.
We used antibodies against Smad2, Smad3, and Smad4 to conduct chromatin immunoprecipitation (CHIP) experiments and qPCR to identify potential binding sites.The results indicated that the location between -1182 to -1032 is the key binding site of Smad2/3/4 complex in HUH7 and HepG2 cell lines (Fig. 4B-G).Above all, these data shows that Smad2/3/4 complex can bind to the TAT gene promotor site and undergo LLPS to regulate TAT gene expression.

Smad2/3/4 complex regulate HCC progression through TAT
Previous study shows that mutation of TAT could promote HCC progression [31].TGF-β signaling pathway could also affect tumor progress [10].Our results showed that Smad2/3/4 could regulate the expression of TAT.We deduced that Smad2/3/4 could regulate HCC progress through TAT.To explore the relationship between TAT and Smad2/3/4 complex in HCC progression.We used CCK and clone formation assays to test the cell proliferation ability.The CCK assay results showed that Smad2/3/4 complex could inhibit hepatoma cell proliferation (Fig. 5A-C, Supplementary Fig. S2A-C).So as the clone formation assay results (Fig. 5D, Supplementary Fig. S2D).Overexpression of TAT could also inhibit HUH7 and HepG2 cell proliferation, downregulation of Smad2 could rescue the phenomenon, furthermore, the results of Smad3 and Smad4 were similar with Smad2 (Fig. 5E-G, Supplementary Fig. S2E-G).The tendency of clone formation assay results was the same as CCK assay results (Fig. 5H-J, Supplementary Fig. S2H-J).
In nude mice tumor formation model, the tumor grew faster when breaking the Smad2/3/4 complex formation.We knocked down Smad2, Smad3 and Smad4, respectively.The tumor volume was obviously increased (Fig. 6A-F), so as the tumor weight (Fig. 6G-I).We use tumor tissues taken form the nude mice tumors to test the TAT expression level and tyrosine level.The results showed that knockdown Smad2, Smad3 or Smad4 could decrease the expression level of TAT (Fig. 6J-L).These results suggest that Smad2/3/4 complex regulates tumor formation directly through regulating TAT.
Fig. 8 Working Model.The TGF-β signaling pathway is activated by TGF-β binding to TβRII, which recruits and phosphorylates TβRI, and then TβRI phosphorylates Smad2 and Smad3, which assemble heterodimeric and trimeric complexes with Smad4.Smad2/3/4 complexes translocate to the nucleus.In the nucleus, Smad2/3/4 complex could undergo LLPS to activate TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis to inhibit HCC progression.The graph was created with BioRender.comSimilar phenomena were observed in Smad3 and Smad4 (Fig. 6G-I, Supplementary Fig. S3G-I).We discovered that overexpressed TAT could increase the expression of cleaved caspase-9, but knocking down Smad2 could decrease cleaved caspase-9 level in hepatoma cell lines, so as Smad3 and Smad4 (Fig. 7J-L, Supplementary Fig. S3J-L).All these results suggest that Smad2/3/4 complex could active caspase-9 and induce hepatoma cell apoptosis through regulating TAT.
Taking together, our research explored that Smad2/3/4 complex could undergo LLPS and binds to TAT gene promotor site to upregulate TAT expression, which can active caspase-9 to induce hepatoma cell apoptosis (Fig. 8).

Discussion
Numerous studies have shown that the accumulation of cellular and molecular aberrations can lead to the development of HCC, including abnormalities in epigenetics, transcriptome, proteome and metabolome [33].Obviously, HCC has remarkable molecular heterogeneity including many genetic and protein-level changes.Therefore, the search for new targeted genes in HCC patients are expected to provide new directions to explore the mechanisms of HCC, as well as new drug targets.
TGF-β signaling pathway plays a dual role in cancer progress, not only inhibits tumor progress by inducing cell cycle arrest, but also induces tumor migration and stimulates epithelial to mesenchymal transition [10].Activation of TGF signaling pathway dependent on receptor TGFβ receptors, but TGFβ receptors (TβR II and TβR I) behave very differently in HCC and other signaling pathway.TβR II mutation occurs in many cancers, such as colon, gastric, pulmonary, ovarian, esophageal, carcinomas [34].TβR I and TβR II often down-regulated in lung, gastric, prostate, and bladder cancers, and TβR I promoter often methylated in gastric cancer [11].TGFβ receptors also play significant roles in stromal cells in the tumor microenvironment.TGF-β showed immunosuppressive effects on both innate and adaptive immune cells [10].The down expression and mutated of TβRII also fund in HCC [7,8], but TβR II and TβR I also related with fibrogenesis and carcinogenesis (fibro-carcinogenesis) in HCC.During hepatitis virusrelated chronic liver diseases, alterations additively shift hepatocytic Smad phospho-isoform signaling from tumor suppression to fibro-carcinogenesis, thereby accelerating liver fibrosis and increasing risk of HCC [35].So, TGF-β signaling pathway showed complexity function in tumor progression.We supposed that Smad2/3/4 could regulate different gene expressions then regulate HCC progress.In our research, Smad2/3/4 complex played a suppression role in HCC through regulating TAT expression.
LLPS is a newly found phenomenon with a variety of possible functions, such as transcriptional or translational regulation [36][37][38], RNA metabolism [39], and signal transduction [40].LLPS can locally concentrate molecules in condensates to activate reactions and signaling processes, increasing the local concentration of key enzymes or protein complexes to accelerate biochemical reactions [17].Interestingly, we analysed the amino acid sequence characters of Smad2, Smad3 and Smad4.The data showed that Smad4 had large amounts of disorder sequence and PrLD domain, which could drive protein to undergo LLPS [36,41,42], suggesting that Smad4 could undergo phase separation.Further, we discovered Smad4 could form liquid droplets at different concentration, and the formation of liquid droplets could be affected by temperature and NaCl concentration.The mixed Smad2, Smad3 and Smad4 solution could also form liquid droplets.All these results showed that Smad2/3/4 complex could undergo phase separation.Previous studies show that Smad2, Smad3 and Smad4 are phosphorylated when TGF-β signaling pathway is activated [10].So, Smad2/3/4 complex must stay on phosphorylation status when the complex translocates into the nucleus and regulates target gene expression.Phosphorylation and methylation are important for protein undergoing LLPS [43][44][45][46].In our research, we used E. coli to express Smad2/3/4 recombinant protein.
However, E. coli system could not express recombinant protein with phosphorylation.Therefore, we could not get the detailed information of the phosphorylation effect on Smad2/3/4 LLPS.We are trying to use the HEK293T cell to express Smad2/3/4 recombinant proteins with phosphorylation modification.This may give us more information about the Smad2/3/4 LLPS.Smad family members not only include Smad2, Smad3 and Smad4, but also contain Smad1, Smad5, Smad7, Smad8 and Smad6 [47].We are also interested in whether these proteins can undergo LLPS.
Previous studies showed that transcription factor can undergo LLPS with mediator to activate gene expression [15,16].Our research found that Smad2/3/4 complex could undergo phase separation, so we believed that Smad2/3/4 complex underwent phase separation in transcription activity.We performed a series of experiments to find that Smad2/3/4 complex could regulate TAT expression then inhibit HCC progress.It is reported that Smad3 and Smad4 can bind directly to DNA(Smad2 can't), and the affinity between Smad3/4 and DNA is relatively weak and need another transcription factor to regulate gene expression [48,49].We are also interested in whether other proteins take part in Smad2/3/4 complex undergoing LLPS.We conducted Smad3 and Smad4 CoIP experiment and mass spectrometry analysis (data not shown), hoped to get more information about this.
Studies have shown that activation of TAT in the tyrosine metabolic pathway influences the treatment resistance of glioblastoma core [50].The TAT gene encodes the mitochondrial protein tyrosine aminotransferase present in liver, which breaks down tyrosine into p-hydroxyphenylpyruvate.Although aberrant expression of TAT in HCC has been shown to be associated with a poor prognosis [31], currently, there is little evidence on how tyrosine metabolism affects cancer progress.To find the cause of the low expression of TAT in HCC, we focused on the TGF-β/Smad2/3/4 signaling pathway, which also acted as a tumor suppressor in cancer [51,52].Our data connected TGF-β signaling pathway and TAT in HCC progress, which brought new insights in TGF-β signaling pathway function in HCC progress.
Above all, we found that Smad2/3/4 could liquid-liquid phase separation to active TAT expression.We experimentally verified a significant link between TAT and Smad2/3/4 complex in HCC progress.In addition, we found that Smad2/3/4 complex could active caspase-9 to induce hepatoma cell apoptosis through regulating TAT.This result contributes to deeper understanding of the role of TGF-β signaling pathway in HCC progress.Meanwhile, it also brings new insights into the mechanism of hepatocellular carcinoma.

Conclusions
Our results showed that TAT showed low expression in clinical HCC samples.Notably, TAT was found to be a novel target gene of Smad2/3/4 complex, and Smad2/3/4 could undergo phase separation to active TAT gene expression.Smad2/3/4 activated caspase-9 through regulating TAT to induce hepatoma cell apoptosis.Our research suggested that inhibiting Smad2/3/4 LLPS might become a new strategy to inhibit HCC progress.Above all, we found that Smad2/3/4 complex could undergo liquid liquid phase separation to active TAT gene expression, and active caspase-9 to induce hepatoma cell apoptosis through regulating TAT to inhibit HCC progress.RC220177), the Screening of small-molecule tumor suppression compounds project (No.SDUZHHT〔2022〕2448) to Zhe Yang.

Fig. 1
Fig. 1 The mRNA level of TAT is positiv with Smad2 and Smad3.A Verification of the mRNA expression levels of TAT.Red represents tumor tissue, black represents normal tissue.B RFS and univariate COX analysis of TAT.C The ROC curve of TAT in HCC.D Expression of TAT in LIHC based on tumor grade.E Expression of TAT in LIHC based on individual cancer stages.F Expression level of TAT in HCC tissues and paracancerous n = 28.G-H The mRNA expression level relationship of TAT and Smad2, Smad3 in HCC tissues, n = 20.P < 0.05, *; P < 0.01,**; P < 0.001, ***

Fig. 2 Fig. 2 (Fig. 3
Fig.2Smad2/3 complex upregulation TAT in HCC cell lines.A-B Relative mRNA and protein levels of TAT were detected by qPCR and western blot analysis in HUH7 which knockdown (A) or overexpress (B) Smad2.C-D Relative mRNA and protein levels of TAT were detected by qPCR and western blot analysis in HepG2 cells which knockdown (C) or overexpress (D) Smad2.E-F Relative mRNA and protein levels of TAT were detected by qPCR and western blot analysis in and HUH7 cells which knockdown (E) or overexpress (F) Smad3.G-H Relative mRNA and protein levels of TAT were detected by qPCR and western blot analysis in HepG2 cells which knockdown (G) or overexpress (H) Smad3.I-J Relative mRNA and protein levels of TAT were detected by qPCR and western blot analysis in HUH7 cells which knockdown (I) or overexpress (J) Smad4.K-L Relative mRNA and protein levels of TAT were detected by qPCR and western blot analysis in HepG2 cells which knockdown (K) or overexpress (L) Smad4.3μg and 5 μg is represented the quality of overexpression vector in each hole of 6-Wells plats.TGF-β1 (150 pM) was added in medium to activate Smads.P < 0.05, *; P < 0.01,**; P < 0.001,***; P < 0.0001, **** (See figure on next page.)
Target vector was transfected into E. coli and cultured in LB medium.Add IPTG to final concentration 0.24 mg/ml when medium OD value to 0.6-0.8.Continuing cultivate the E. coli for 16-24 h in 16-24 ℃, then collect the E. coli.Treat the bacteria with ultrasonic crusher (200W, 3 s on and 4 s off ).Follow the protocol of Ni-NTA column to purify protein.